In mammals and insects, intercellular signaling by peptides and protein hormones is critical for the functions of circuits regulating sleep and circadian behavior. In this application, we propose experiments to test the hypothesis that neuronal secretion and action of a novel immunoglobulin (Ig)-domain protein is required for proper function of the Drosophila circadian and sleep circuits. Ig superfamily molecules are important mediators of neural development, synapse formation, plasticity and injury (1;2), although less is known about the adult functions for these proteins. In recent expression profiling studies, we identified numerous Drosophila Ig protein-encoding genes that are expressed in the adult brain. Sixteen of them, including the CG141441 (Nkt) gene, have high adult nervous system expression and encode Ig proteins that are predicted to be secreted molecules. (FlyAtlas; (3)). In unpublished studies, we have shown that flies with RNAi-generated pan-neuronal deficits for Nkt exhibit reduced nighttime sleep and lengthened circadian period. Selective knockdown of Nkt in clock neurons causes a lengthened circadian period with no effect on sleep. These results suggest that Nkt is required in different neuronal cell types for th regulation of sleep and circadian behavior. Interestingly, genetic interaction with a period (pers) mutation suggests that NKT acts on pacemaker neurons and through the molecular clock to regulate circadian period. As NKT is predicted to be a small secreted Ig protein, we hypothesize that it serves a signaling function, similar to PDF neuropeptide, in the circadian circuit. In this R21 application, we propose to study the functions of NKT in the regulation of adult rhythmic behavior and sleep. Aim 1 will use conditional expression approaches to verify that NKT protein has a physiological requirement in the adult brain for the regulation of behavior. Additionally, it will delineate the neuronal requirements for the protein in sleep and circadian behavior. Aim 2 will examine genetic interactions with clock and sleep-altering mutations to better understand NKT's mechanistic functions in the relevant neuronal circuits. This aim will also ask whether NKT is secreted from neurons and test the hypothesis that the Ig protein is required for molecular oscillator function or synchronization of the clock neuron population. Proteins homologous to NKT, with single immunoglobulin domains are present in many species including C. elegans, Drosophila and humans (e.g., small secreted Neuregulin-2 isoforms). Thus, the research is likely to provide novel insights about signaling mechanisms that function in the differentiated brain.